Autophagy dictates sensitivity to PRMT5 inhibitor in breast cancer

Protein arginine methyltransferase 5 (PRMT5) catalyzes mono-methylation and symmetric di-methylation on arginine residues and has emerged as a potential antitumor target with inhibitors being tested in clinical trials. However, it remains unknown how the efficacy of PRMT5 inhibitors is regulated. Here we report that autophagy blockage enhances cellular sensitivity to PRMT5 inhibitor in triple negative breast cancer cells. Genetic ablation or pharmacological inhibition of PRMT5 triggers cytoprotective autophagy. Mechanistically, PRMT5 catalyzes monomethylation of ULK1 at R532 to suppress ULK1 activation, leading to attenuation of autophagy. As a result, ULK1 inhibition blocks PRMT5 deficiency-induced autophagy and sensitizes cells to PRMT5 inhibitor. Our study not only identifies autophagy as an inducible factor that dictates cellular sensitivity to PRMT5 inhibitor, but also unearths a critical molecular mechanism by which PRMT5 regulates autophagy through methylating ULK1, providing a rationale for the combination of PRMT5 and autophagy inhibitors in cancer therapy.


Deficiency in PRMT5 induces autophagosome formation.
To explore whether PRMT5 inhibition induces cytoprotective autophagy, we knocked out PRMT5 using CRISPR/Cas9 gene editing in multiple breast cancer lines and evaluated autophagy activity. Strikingly, depletion of PRMT5 led to an elevation of LC3-II/I ratio and a reduction of p62 protein levels under normal culture condition ( Fig. 2a and Supplementary Fig. 2a,  b). Consistently, treatment of cells with GSK3326595 increased autophagy activity ( Fig. 2b and Supplementary  Fig. 2c). To further support these immunoblot results, we monitored autophagy activity using the GFP-LC3 report system [52] and found that there was a significant increase of GFP-LC3 puncta in GSK3326595-treated cells (Fig. 2c, d). Consistently, cells expressing the enzymatically dead mutant PRMT5-E444Q [53] also enhanced LC3-II accumulation and p62 degradation, compared to cells expressing PRMT5-WT ( Supplementary Fig. 2d), suggesting that PRMT5 regulates basal autophagy in a enzymatic-dependent manner.
Given that autophagy is a key biological process for adaptation to various stress events, such as nutrient deprivation, we next investigated whether PRMT5 is also involved in stress-induced autophagy. To this end, we found that compared to control cells, PRMT5-depleted cells displayed an additive LC3-II accumulation in response to the starvation of amino acids ( Supplementary Fig. 2e). In contrast, overexpression of the PRMT5-E444Q mutant enhanced autophagy in the absence of amino acids, compared to overexpression of PRMT5-WT ( Supplementary Fig. 2f). Previous studies have demonstrated that mTORC1-mediated phosphorylation of ULK1 at S757 is a key switch of autophagy induction in response to stresses [54,55] . Interestingly, we did not observe a significant difference on phosphorylation of ULK1-S757 between PRMT5-WT and PRMT5-E444Q expressing cells ( Supplementary Fig. 2f). These results indicate that PRMT5-mediated regulation of autophagy is likely independent of the mTORC1 pathway and has an additive effect on nutrient deficiency-induced autophagy.
Of note, the accumulation of LC3-II in PRMT5-deficient cells could be caused by either enhanced LC3-I conversion to LC3-II or impaired LC3-II degradation [56] . To distinguish these two scenarios, we measured the autophagic flux using the mRFP-GFP-LC3 reporter system, which is based on the principle that GFP, but not mRFP, is quenched in the acidic environment, such as lysosome [57] . An increase of yellow (RFP + /GFP + ) and red (RFP + ) puncta indicates enhanced autophagosome formation, while only accumulation of yellow puncta suggests impairment in autophagosome-lysosome fusion and degradation. Notably, a significant accumulation of both yellow and red puncta of LC3 was observed in PRMT5-depleted cells (Fig. 2e, f). Moreover, treatment of cells with chloroquine (CQ), which inhibits autophagic flux by blocking autophagosome-lysosome fusion [50] , led to a further accumulation of LC3-II in PRMT5-depleted cells (Fig. 2g). These results suggest that PRMT5 suppresses autophagosome formation, but not autophagosome-lysosome fusion. www.nature.com/scientificreports/ ULK1 is required for PRMT5-mediated regulation of autophagy. To investigate whether autophagosome formation induced by PRMT5 deficiency depends on the canonical autophagy pathway, we genetically ablated the core ATG genes involved in the initiation and nucleation stages (Fig. 3a). Strikingly, depletion of ULK1 largely blocked the induction of LC3-II in GSK3326595-treated or PRMT5-depleted cells (Fig. 3b, c). Moreover, ablation of ATG13, a component that enhances ULK1 activity and stability [38] , phenocopied the effects of ULK1 depletion ( Supplementary Fig. 3). Furthermore, depletion of Beclin 1 that mediates nucleation downstream of ULK1 led to the blockage of autophagy induced by PRMT5 inhibitor or PRMT5 depletion (Fig. 3d, e). We also confirmed the immunoblot results using GFP-LC3 system and found that ULK1   www.nature.com/scientificreports/ depletion strongly decreased the formation of GFP-LC3 puncta in PRMT5-depleted cells (Fig. 3f, g). These results suggest that PRMT5 regulates autophagy in part through ULK1.
PRMT5 interacts and methylates ULK1 at Arg532. A recent study on ULK1 interactome identified PRMT5 as a partner of ULK1 [58] . We speculated that this interaction plays a role in PRMT5-mediated regulation of autophagy. Consistent with the proteomic study [58] , we found that PRMT5 specifically co-immunoprecipitated endogenous ULK1, but not Beclin 1 (Fig. 4a). Reciprocally, ULK1 interacted with PRMT5, but not PRMT1 ( Fig. 4b and Supplementary Fig. 4a). ULK1 contains an N-terminal kinase domain (KD), intrinsically disordered region (IDR) that is modified by multiple kinases for regulation of ULK1 activation, and a C-terminal early autophagy tethering (EAT) domain that is responsible for recruitment of ATG13, FIP200, and ATG101 [59] . We found that PRMT5 specifically bound to the KD of ULK1 ( Supplementary Fig. 4b), depletion of which abolished their interactions ( Supplementary Fig. 4c). These results demonstrate that the KD is necessary and sufficient for ULK1 binding to PRMT5, which is distinct from ULK1 interaction with its known partners ( Supplementary  Fig. 4d). Next, we investigated whether ULK1 is a substrate of PRMT5. Immunoblot analysis using an antibody against pan MMA [60] showed that ULK1 was monomethylated ( Supplementary Fig. 4e). Overexpression of PRMT5-WT, but not the enzymatically dead mutant PRMT5-E444Q, promoted MMA formation of ULK1 (Fig. 4c). In www.nature.com/scientificreports/ contrast, the MMA levels of ULK1 were severely decreased upon PRMT5 depletion (Fig. 4d). To identify which residue(s) is methylated by PRMT5, we analyzed ULK1 protein sequence by arginine methylation prediction tool, GPS-MSP [61] . Six arginine residues that were ranked top score were selected for further analyses (Fig. 4e).
Notably, the R532K mutation, but not other mutations, abolished PRMT5-mediated MMA formation of ULK1 in cells (Fig. 4f, g). To demonstrate that PRMT5 directly methylates ULK1 at R532, we performed in vitro arginine methylation assays [62] using the recombinant GST-ULK1 truncated protein that encompasses R532 (1-649 aa). Consistent with the finding in cells (Fig. 4g), the R532K mutant largely blocked PRMT5-mediated methylation in vitro (Fig. 4h). Although no SDMA signal was detected by immunoblot using the pan anti-SDMA antibody [60] , we could not rule out SDMA modification on ULK1-R532 because it might not be recognized by this antibody. www.nature.com/scientificreports/ Indeed, we identified dimethylation of ULK1 at R532 by mass spectrometry (Supplementary Fig. 4f). It warrants future development of the antibody that specifically recognizes symmetric dimethylation of ULK1-R532. Taken together, these results demonstrate that PRMT5 is the major physiological methyltransferases responsible for methylation of ULK1 on R532. Interestingly, posttranslational modifications of ULK1, including phosphorylation by mTOR/AMPK [54,63] and acetylation by TIP60 [64] , were generally regulated by stresses. However, neither ULK1 MMA nor interaction between PRMT5 and ULK1 was affected in response to amino acid deprivation ( Supplementary Fig. 4g, h), arguing that PRMT5-mediated regulation of ULK1 is independent of stress, at least nutrient deficiency.
Next, we sought to investigate how ULK1-R532K enhances its kinase activity. Both ULK1-WT and ULK1-R532K bound to FIP200 and ATG13 at a comparable level ( Supplementary Fig. 5b, c), indicating the ULK complex formation was not affected. We also did not observe a change of ULK1-R532K binding to its substrates, Beclin 1 and Ambra 1 ( Supplementary Fig. 5c, d). Moreover, the interaction between ULK1-R532K and AMPK or Raptor (an essential subunit of mTORC1) was not significantly changed, compared to ULK1-WT ( Supplementary  Fig. 5e), further supporting the notion that R532 methylation regulates ULK1 activation is independent of or parallel to the mTORC1/AMPK pathway. These results suggest that ULK1-R532 methylation impairs its kinase activity unlikely through modulating ULK1 interactions with its partners. www.nature.com/scientificreports/ ULK inhibitor sensitizes resistant TNBC cells to PRMT5 inhibitor. Since ULK1 is a key druggable serine/threonine kinase for the induction of cytoprotective autophagy, targeting ULK1 therefore represents a promising therapeutic strategy for overcoming drug resistance [67] . Having demonstrated that ULK1 plays a critical role in PRMT5-mediated autophagy regulation, we interrogated whether ULK1 inhibition would enhance sensitivity to PRMT5 inhibitor. Treatment with ULK1/2 inhibitor MRT68921 [68] largely suppressed GSK3326595induced autophagy, as evidenced by a decrease of the LC3B II/I ratio and GFP-LC3B puncta ( Fig. 6a and Supplementary Fig. 6a). As a result, combination of MRT68921 with GSK3326595 significantly decreased cell viability and colony formation in TNBC cells, compared to single agent ( Fig. 6b-d). Moreover, apoptosis was strongly enhanced in cells treated with both GSK3326595 and MRT68921, compared to cells treated with single agent (Fig. 6e). Furthermore, cells expressing ULK-R532K displayed more colonies than cells expressing ULK-WT in the presence of GSK3326595 (Fig. 6f, g). These results suggest that ULK1 inhibition suppresses cytoprotective autophagy and consequently confers sensitivity to PRMT5 inhibitor in TNBC cells.

Discussion
Over the past decade, extensive studies suggest that PRMT5 functions as an oncoprotein in various cancers through both epigenetic and non-epigenetic mechanisms [11] . Notably, PRMT5 is overexpressed in more than 50% of primary breast tumors and 70% of metastatic breast tumors, with strongest expression in TNBC [15,29] . These findings make PRMT5 as an attractive therapeutic target and pharmacological inhibition of PRMT5 represents a promising strategy for cancer therapy [69] . Our study demonstrates that PRMT5 inhibition evokes cytoprotective autophagy in part through promoting ULK1 activation, which sustains cell survival and confers resistance to PRMT5 inhibitors, and blockage of autophagy with ULK1 inhibitor or CQ remarkedly improve the efficacy of PRMT5 inhibitor in TNBC (Fig. 6h). Thus, our data establish a foundation for treatment of breast cancer with combinatorial inhibition of PRMT5 and autophagy. PRMT5 is a versatile protein involved in many cellular processes [70] . Our finding revealed autophagy as another cellular process regulated by PRMT5. Although we showed that PRMT5 directly methylates ULK1 at R532 to suppress its kinase activity and basal autophagic function, we agree that ULK1-R532K mutant does not fully recapitulate the levels of autophagy induced by PRMT5 inhibition. It is possible that other mechanisms also contribute to PRMT5-mediated regulation of autophagy. For example, other ATG proteins and upstream autophagy modulators could be putative substrates of PRMT5. Indeed, PRMT5 have been documented to methylate and enhance AKT activation [25] , which negatively regulate autophagy by phosphorylating Beclin 1 [71] . Moreover, PRMT5 is a crucial player in DNA damage response and DNA repair [72] , deficiency in which can induce autophagy [73] . These mechanisms may synergize with the defect in ULK1-R532 methylation to boost autophagy under condition of PRMT5 inhibition. www.nature.com/scientificreports/ ULK1 functions as a conserved serine/threonine kinase in the autophagy pathway to sense upstream signals and initiate autophagy. During this process, PTMs, particularly phosphorylation, play a critical role in the dynamic regulation of the ULK1 activity [74] . Notably, by phosphorylating ULK1 at distinct residues of IDR, mTORC1 inhibits while AMPK activates autophagy in response to the changes of nutrition or energy in cells [54,63] . Our study demonstrates that PRMT5-mediated methylation of ULK1 at R532 reduces its kinase activity, adding another layer of ULK1 regulation regardless of the availability of nutrition. However, except for ubiquitination that has been shown to directly affect ULK1 stability [75] , the detailed mechanisms underlying how PTMs affects ULK1 activation have not yet been clearly established. Similarly, although it moderately affects ULK1 interaction with some of its substrates, ULK1-R532 methylation may also control ULK1 activity through other mechanisms. For example, ULK1-R532 methylation may cause its structurally conformational change or its interactions with other regulators, which warrants further studies.
While this manuscript was being prepared, a study reported that PRMT5/KDM5C-mediated dimethylation of ULK1 at R170 activates ULK1 to induce autophagy in LN229 glioblastoma (GBM) cells, Huh7 hepatocellular carcinoma (HCC) cells, and human oral keratinocytes (HOKs) in hypoxic environment, but not in normoxic condition [76] . However, it is unclear whether R170 is the sole site methylated by PRMT5 because they detected ULK1 arginine methylation only using the anti-ULK1-R170me2s antibody. Moreover, it is still needed to determine whether PRMT5 is involved regulation of autophagy under normoxic condition. By using the radioisotopebased in vitro arginine methylation assay, we demonstrated that R532 is the major methylation site by PRMT5. Our data also showed that PRMT5 depletion or PRMT5 inhibitor significantly induced autophagy in TNBC cells cultured in normal conditions. Therefore, PRMT5-mediated regulation of ULK1 activation and autophagy induction is likely dependent on environments and cell types.
Antibodies. All primary antibodies were diluted with 5% non-fat milk in TBST buffer for Western blot. www.nature.com/scientificreports/ amounts of whole cell lysates were resolved by SDS-PAGE and immunoblotted with indicated antibodies. For IP, 2000-5000 μg lysates were incubated with agarose conjugated antibodies for 3-5 h at 4 °C. Immunoprecipitants were washed three times with NETN buffer (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA and 0.5% NP-40) or Triton buffer before being resolved by SDS-PAGE. Anti-HA agarose beads (A2095) and anti-FLAG agarose beads (A2220) were purchased from Sigma-Aldrich. Anti-Myc agarose beads (658502) were purchased from BioLegend. Some blots were cut prior to hybridization with primary antibodies, but one full-length original, unprocessed blot for each antibody was provided in the Supplementary Materials.
Purification of GST-tagged protein from E. coli. Recombinant  The samples were resolved by SDS-PAGE and transferred to PVDF membrane, which was then sprayed with EN3HANCE (Perkin Elmer) and exposed to X-ray film.
In vitro kinase assays. 3  Immunofluorescence staining. Cells grown on glass coverslips were fixed with 4% paraformaldehyde for 15 min at room temperature, washed three times with PBS, and then permeabilized with 0.05% Triton X-100 for 10 min at room temperature. Following three washes with PBS, cells were stained with DAPI, washed four times with PBS and mounted using vibrance antifade mounting medium (Vector Laboratories, H-1700). Images were taken by Leica SP8 Confocal microscope and puncta were counted manually.
Mass spectrometric analysis of ULK1-R532 methylation. HEK293T cells were transfected with HA-ULK1. Forty-eight hours post transfection, the cells were lysed in Triton buffer, followed by immunoprecipitation. The immunoprecipitates were resolved by SDS-PAGE and visualized using GelCode blue staining reagent (Thermo Scientific, 24590). The protein band containing HA-ULK1 was excised and digested with trypsin. Peptides were analyzed on an EASY nLC 1200 in-line with the Orbitrap Fusion Lumos Tribrid mass spectrometer (ThermoScientific). Peptides were pressure loaded at 800 bar and separated on a C18 reversed phase column (Acclaim PepMap RSLC, 75 μm × 50 cm (C18, 2 μm, 100 Å)) (Thermo Fisher) using a gradient of 2-35% B in 180 min (Solvent A: 0.1% FA; Solvent B: 80% ACN/0.1% FA) at a flow rate of 300 nL/min at 45 °C. Mass spectra were acquired in datadependent mode with a high resolution (60,000) Fourier Transform mass spectrometry (FTMS) survey scan followed by MS/MS of the most intense precursors with a cycle time of 3 s. The automatic gain control target value was 4.0e5 for the survey MS1 scan. Precursors were isolated with a 1.6 m/z window with a maximum injection time of 50 ms. Tandem mass spectra were acquired using higher-energy collisional dissociation (HCD) and electron transfer dissociation (ETD) for each peptide precursor in an alternating fashion. The HCD collision energy was 35% and ETD was performed using the calibrated charge dependent ETD parameters. The fragment ions were detected in the Orbitrap at 15,000 resolution. Spectra were searched against a custom database containing human ULK1 and a database of common contaminants using MaxQuant and Proteome Discoverer. The false discovery rate, determined using a reversed database strategy, was set at 1% at the peptide and modification site levels. Fully tryptic peptides with a minimum of seven residues were required including cleavage between lysine and proline. Two missed cleavages were permitted. Sites of modification were manually verified.
Cell viability assays. Cells were seeded in 96-well plate at 500-1000 cells per well for 24 h and then treated with indicated doses of inhibitors for 4 days. Cell viability was determined using the Cell Titer-Glo cell viability assay kit according to the manufacturer's instructions (Promega, G7570).
Clonogenic survival assays. Cells were seeded in 6-well plates at 300-500 cells per well for 24 h and then treated with indicated inhibitors for 8-10 days until visible colonies formation. Fresh medium with inhibitors was replaced every 3 days. Colonies were fixed with 10% ethanol and 10% acetic acid for 30 min and then stained with 0.4% crystal violent in 20% ethanol for 30 min, followed by wash with dH2O and manually counted.